Method of reducing the number of microorganisms in a media and a method of preservation

ABSTRACT

What is disclosed is a method of reducing microorganisms by contacting the microorganisms with the surface of a substrate which has been altered by contacting a substrate which develops a negative charge in water with a substance that ionizes in water to form cations and anions, which cations absorb to the substrate by a process of ion exchange in which protons are replaced by the cations of the ionizing substance. The altered surfaces can include hospital sheeting, clothing and a wide variety of surfaces which come into contact with microorganisms.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part application of Ser. No.19,532, filed Mar. 12, 1979 now abandoned.

Almost concurrently with the notion that microorganisms cause infectionand disease came the discovery that the microorganisms could becontrolled either by killing all of them or reducing their numberssignificantly.

Many methods have been devised that would accomplish the end resultincluding reducing all known infected things to ashes. As a practicalmatter, because all such infected things could not be summarilysubjected to such treatment, other, more specific methods were devisedto control the microorganisms. One such specific method was thetreatment of milk and similar products, at elevated temperatures highenough to destroy objectional organisms but not high enough to alter thechemical characteristics of the milk. This method was developed by LouisPasteur during the mid-nineteenth century. Sterilization wherefoodstuffs were not involved constituted the use of chemicals such asphenol. Phenol is effective but is a very harsh chemical in terms of itsacidic properties. Similar microbial poisons have been commerciallyavailable for a number of years. Certain amines and their quaternarysalts, boric and carboxylic acids and their salts and a host of otherchemicals have been used. These materials were used neat or in aqueoussolutions and then placed on the substrate to be sterilized.

The utility and commercial potential of bacteriostatic properties ofcertain cationic nitrogen compounds is well known and seems first tohave been recognized about 1935 in U.S. Pat. No. 2,108,765 and, G.Domagk, "A New Class Of Disinfectant Materials", Deut. Med. Wochn., 61,829 (1935). Domagk recognized that if at least one group in an amine wassufficiently large and properly hydrophobic to give the moleculesurfactant properties in water, such a molecule then had bacteriostaticor bacteriocidal properties. Following these publications, scores ofcationic surfactants were found to have antimicrobial properties, andthe teaching of Domagk was proved to be correct.

It is observed that surfactant properties and antimicrobial activity arefound in common among compounds with at least one positively chargednitrogen atom and at least one hydrophobic group. The hydrophobic groupshould be equivalent to an aliphatic group of at least C₈ H₁₇ - and notlarger than C₂₂ H₄₅ -.

An excellent review of the antimicrobial behavior of cationic substancesin solution is to be found in "Cationic Surfactants", Marcel Dekker,Inc., New York, 1970, especially Chapter 14, Carl A.Lawrence-"Germicidal Properties of Cationic Surfactants"; and Chapter11, Martin E. Ginn, "Adsorption of Cationic Surfactants on MiscellaneousSolid Substrates".

Researchers have investigated ways in which substances which were provento be useful antimicrobials in solution could be made more persistent onsubstrates. One such way was to attach an antimicrobial agent to asubstrate by some means.

In one method, an antimicrobial agent was mixed with a curable coating.This method was quite satisfactory for prolonging the residence of theantimicrobial agent but problems were still encountered as theantimicrobial agent leached from the coating and was washed away.

A better method evolved around 1965 when researchers discovered thatantimicrobial organofunctionalsilanes could be chemically bound tocertain substrates by what were believed to be Si-O linkages. The methodwas described as orienting the organofunctional silane in such a waythat hydrolyzable groups on the silicon atom were hydrolyzed to silanolsand the silanols formed chemical bonds with the substrate and theantimicrobial end of the molecule, for example a quaternary nitrogen,was oriented away from the substrate; P. A. Walters, E. A. Abbott and A.J. Isquith, Applied Microbiology, Feb. 1973, pages 253-256. Theoriesabiding at the time alleged that any molecule having a reactive silylgroup to bind to a surface and which also displayed antimicrobialactivity in solution would function this way. In other words, if themolecule had activity in solution, this activity could be fixed to asurface by means of the silanol groups. The fixation would be so strongthat little or no material would be removed from the surface by water.Thus, such a surface would affect microorganisms in water without lossof the active structure from the surface.

The theory could not be substantiated because no method existed by whichthe theory could be tested. The methods available were designed tomeasure the activity of an agent in solution, and this fact is importantbecause the present invention deviates significantly from that theoryand greatly increases the number and variety of substances that can beuseful to render surfaces antimicrobial.

A clear statement of the theory that a totally bound substance could notaffect microorganisms can be found in L. A. Vol'f, "ImpartingAntimicrobial Properties to Fibers", Tekstilnaya Promnyshlenmost No. 8,9 (1965), U.S.S.R. (Translation by Techtron Corp.), who stated,"Imparting antimicrobial activity to fibers can be carried out in twoways: (1) by sorption of a chemotherapeutic preparation or antisepticagent (with subsequent desorption) and (2) by chemical linking of thefiber with compounds which impart to the fibers an anti-bacterial effect(with their subsequent cleavage)." Speaking of method (2) Vol'f goes onto state, "The problem of the fixation of antimicrobial substances onthe fiber by means of a chemical bond contains at first sight, twocontradictory postulates. Actually on the one hand it is dictated by theeffort to prevent the removal of groupings which are responsible for theantimicrobial effect from the fiber during use and on the other hand itis necessary to assume the transportation (diffusion) of these groupswithin the microbial cells for otherwise, the main goal of destroyingthe pathogenic microflora at a distance will not be achieved." He alsoadds, ". . . the necessary condition for the manifestation ofantibacterial action is the presence of moisture. It is precisely thiswhich accomplishes the transport of active ingredients to themicroorganisms."

In 1967, Canadian Pat. No. 774,529 was issued and was completelycontrary to Vol'f's opinion. This patent stated that antimicrobialactivity could in fact be put on a substrate by binding an antimicrobialorganosilicon compound to a substrate but this patent showed no exampleof how this should be done, nor an example that this had been done. Thefirst example in which this was thought to be demonstrated was taughtclearly by Isquith et al. later in a publication; A. J. Isquith, E. A.Abbott and P. A. Walters, Applied Microbiology, December, 1972, Pages859-863. In this publication they taught that only cationicantimicrobial agents which were substituted by a (CH₃ O)₃ Si- groupcould display antibacterial activity when deposited on a surface.

P. A. Walters, E. A. Abbott and A. J. Isquith, Applied Microbiology, 25,No. 2, p. 253-256, Feb. 1973 amplified this teaching. They taught that3-(trimethoxysilyl)propyldimethylalkylammonium chlorides, with alkylchain lengths from 6 to 22 carbons in solution were capable of killingalgae. They state, "these compounds retained biological activity whendurably attached to a surface". They further teach that this was a"unique property" of these compounds and that such closely similarcompounds as the commercial Benzalkonium chlorides (WinthropLaboratories, Division of Sterling Drug Inc., New York, NY) did notdisplay this property.

The teaching was further amplified by E. A. Abbott and A. J. Isquith,U.S. Pat. No. 3,794,736, Feb. 26, 1974. In this patent they teach that avariety of siloxane-substituted amines and amine salts which displayantibacterial and antifungal properties in solution, continue to displaythese properties when the substances were deposited on surfaces. Theyclaimed a method of inhibiting growth of bacteria and fungi bycontacting said organisms with a surface that had been coated with avariety of siloxane-substituted organic amines or their salts.

One substance favored by these investigators has been adopted forcommercial use to prepare antimicrobial surfaces. This substance isessentially a methanol solution of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₃ C₁₈ H₃₇ ⁺Cl⁻.

The use of a filler, e.g. silica or titanium dioxide, which had beentreated with an organosilicon-substituted quaternary ammonium salt, toinhibit the growth of micro-organisms in or on the surface of apolymeric organic matrix into which the filler had been incorporated wastaught in Canadian Pat. No. 1,010,782, issued May 24, 1977, to Roth.

This is a new way to use a surface treated as taught by Isquith, et al.and Roth was surprised to discover that a filler treated with 10% byweight of (CH₃ O)₃ Si(CH₂)₃ NC₁₈ H₃₇ (CH₃)₃ ⁺ Cl⁻ was effective,unexpectedly contrary to the teaching of Isquith, et al. in that thissubstance is not antimicrobial in solution.

Thus, it can be observed that all prior art indicates that antimicrobialsystems can be prepared by (1) treatment of surfaces with harshchemicals; (2) chemical bonding of chemical agents to substrates.

The Invention

It has now been discovered that there is a significantly better way toreduce the number of viable microorganisms. By changing the surfacecharacteristics of a substrate and then contacting microorganisms withthe changed surface of the substrate, the microorganisms are killed.

Thus what is disclosed is a method of reducing the number of viablemicroorganisms in media by physically contacting the microorganisms witha surface which has been altered in a manner which comprises contactinga substrate, which develops a negative charge in water, with a substancethat ionizes in water to form cations and anions, which substanceconsists of organic amines having the formula R¹ R² R³ N in which R¹, R²and R³ are independently hydrogen, alkyl or aralkyl groups wherein thereis a total of less than 30 carbon atoms in the molecule; an organicquaternary ammonium salt of the formula R⁴ R⁵ R⁶ R⁷ N⁺ X⁻ wherein R⁴,R⁵, R⁶ and R⁷ are independently alkyl or aralkyl groups wherein there isa total of less than 30 carbon atoms in the molecule and X⁻ is a watersoluble monovalent anion; a sulfonium salt of the formula R⁸ R⁹ R¹⁰ S⁺X⁻ in which R⁸, R⁹, and R¹⁰ are independently alkyl groups or aralkylgroups wherein there is a total of less than 30 carbon atoms in themolecule or a silylsubstituted alkyl radical of the formula ##STR1## inwhich Y is a hydrolyzable group, Q is an alkyl radical of 1 or 2 carbonatoms of (CH₃)₃ SiO, a has an average value of 0-3; b has an averagevalue of 0-3, n is an integer of 1 or greater and X⁻ is a water solublemonovalent anion; an isothiuronium salt of the formula RSC(NH₂)₂ ⁺ X⁻ inwhich R is independently alkyl or aralkyl groups wherein there is atotal of less than 20 carbon atoms in the molecule or a silylsubstituted alkyl radical of the formula ##STR2## in which Y is ahydrolyzable group, Q is an alkyl radical of 1 or 2 carbon atoms or(CH₃)₃ SiO, a has an average value of 0-3; b has an average value of0-3, and n is an integer of 1 or greater and X⁻ is a water solublemonovalent anion; a phosphonium salt of the formula R⁴ R⁵ R⁶ R⁷ P⁺ X⁻ inwhich R⁴, R⁵, R⁶ and R⁷ are independently alkyl groups or aralkyl groupswherein there is a total of less than 30 carbon atoms in the molecule ora silyl substituted alkyl radical of the formula ##STR3## in which Y isa hydrolyzable group, Q is an alkyl radical of 1 or 2 carbon atoms or(CH₃)₃ SiO, a has an average value of 0-3; b has an average value of0-3, n is an integer of 1 or greater and X⁻ is a water solublemonovalent anion; a sulfonium salt of the formula O[Si(CH₃)₂ C_(d)H_(2d) S⁺ (R¹¹)₂ X⁻ ]₂ in which R¹¹ is independently an alkyl group oraralkyl group wherein there is a total of less than 60 carbon atoms inthe molecule, d is an integer of 1 or greater and X⁻ is a water solublemonovalent anion; an isothiuronium salt of the formula O[Si(CH₃)₂ C_(d)H_(2d) S⁺ C(NH₂)₂ X⁻ ]₂ in which d is an integer of 1 or greater and X⁻is a water soluble monovalent anion; a phosphonium salt of the formulaO[Si(CH₃).sub. 2 C_(d) H_(2d) P⁺ (R¹²)₃ X⁻ ]₂ in which R¹² isindependently an alkyl group or aralkyl group wherein there is a totalof less than 60 carbon atoms in the molecule, d is an integer of 1 orgreater and X⁻ is a water soluble monovalent anion and, an organic amineof the formula ##STR4## in which d is an integer of 1 or greater.

The value of n is normally 1-10 carbon atoms. For all practicalpurposes, d generally has a value of 1-10 carbon atoms.

As can be observed, the size of the alkyl or aralkyl groups is dependenton the type of substance that is being utilized. Thus, if the materialis an organic amine of the formula R¹ R² R³ N, a quaternary ammoniumsalt of the formula R⁴ R⁵ R⁶ R⁷ N⁺ X⁻, a sulfonium salt of the formulaR⁸ R⁹ R¹⁰ S⁺ X⁻ or a phosphonium salt of the formula R⁴ R⁵ R⁶ R⁷ P⁺ X⁻,the total number of carbon atoms in the molecule can be as large as 30while the isothiuronium salts of the formula RSC(NH₂)₂ ⁺ X⁻ require atotal of 20 carbon atoms or less. When the substance is a sulfonium saltof the formula O[Si(CH₃)₂ C_(d) H_(2d) S⁺ (R¹¹)₂ X⁻ ]₂ or a phosphoniumsalt of the formula O[Si(CH₃)₂ C_(d) H_(2d) P⁺ (R¹²)₃ X⁻ ]₂, the totalnumber of carbon atoms in the molecule can be as large as 60. It shouldbe understood that as regards the latter sulfonium and phosphoniumsalts, this invention does not contemplate alkyl or aralkyl groups of 60carbon atoms but instead contemplates a disiloxane which contains alkylor aralkyl groups on each sulfur or phosphorus atom such that there areless than 30 carbon atoms per each sulfur or phosphorus per molecule.The size and type of the alkyl or aralkyl group is significant in thatthe larger groups contribute to the permanence of the surface alterationwhich in turn contributes to the permanence of the antimicrobial effect.

Bulky organic cations adsorb very strongly to substrates. If the organicgroups have straight chain alkyl configurations then they tend to layclosely packed, side by side, extending essentially upward from thesubstrate surface. This closer proximity leads to bonding between thealkyl groups through Van der Waals' forces or some similar effect. Thisincreased bonding mechanism tends to enhance the permanence of thecationic surface.

For purposes of this invention, generally one or two large alkyl oraralkyl groups on the molecule are used. For all practical purposes,molecules containing more than two large alkyl or aralkyl groups arehard to prepare and further they tend to be too bulky to give theoptimum bonding between groups necessary for this invention. Preferredfor this invention are those compounds that contain at least one alkylor aralkyl group having greater than 10 carbon atoms.

X⁻ for purposes of this invention is a water soluble monovalent anion.Examples of preferred anions are Cl⁻, Br⁻, I⁻ and CH₃ COO⁻.

The advantages of the newly discovered method are that a very largevariety of surfaces, which include all which develop a negative chargein contact with water, can be altered to have antimicrobial activity bytreatment with a very large variety of substances which need have nobiocidal activity in solution, but which need only to ionize in solutionin water to form a cation other than a proton.

The most critical aspect of this invention is the fact that theinterface between surfaces of the substrates of this invention and watermust be altered. It should be understood that the substrate is notchemically altered. The interface between the surface of the substrateand water is altered in that adsorbed water on the surface is altered.Substrates that fall within the scope of this invention are anysubstrates which develop a negative charge on their surfaces in thepresence of water. Such things as fibers, textiles, particulatematerials such as sand, earth, concrete and masonry surfaces, wood,plastics and polymers all develop negative charges on their surfaces inthe presence of water.

The adsorption of cations upon mineral surfaces from water has beenstudied extensively for many years and a carefully detailed theory ofthe physical chemistry of the process has been developed. The adsorptionof cations upon organic surfaces has also been extensively studied,although the theory in this case is not as completely developed. Theconcept that an electrically charged layer develops at a solid-liquidboundary or interface has been accepted for at least as long as 160years (F. Reuss, 1809, R. Porret, 1816). That a process of ion exchangeoccurs in this charged layer has been believed for at least 100 years(H. von Helmholtz, 1879). A mathematical semi-quantitative descriptionof the process was well developed at least 50 years ago. (O. Stern,1924). Any good textbook on the physical chemistry of surfaces describesthese phenomena. See S. Glasstone, Text-Book of Physical Chemistry, D.Van Nostrand Company, Inc. New York, NY (1940) pp. 1194 et seq.

When surfaces are subjected to solutions of a cationic material as laterdefined in this specification, for example, a cationic surfactant, thesurface is altered to the extent that hydrogen ions of adsorbed waterare removed from the surface and moved to the bulk water and the cationsmove to the surface. It is theorized, but the inventors do not wish tobe held to such a theory, that the cations replace hydrogen ions ofwater adsorbed on the surface of the substrate until the cations arepacked very closely together. Further, if the cationic material alsocontains appropriate groups in the molecule (as explained later in thisspecification), the alteration is permanent and the substrate surfaceremains antimicrobial.

Water adsorbed on the substrate is highly ionized, thus H³⁰ OH⁻. Whensuch a surface is treated with a cationic material, the hydrogen ions ofthe adsorbed water preferentially move to the surrounding bulk water andthe cation of the cationic material moves preferentially to the surface.Thus, it is theorized that there is an equilibrium at the surface i.e.H⁺ OH⁻ +H₂ O⃡HO⁻ +H₃ O⁺. When the cations from the cationic materialapproach the surface the equilibrium is upset and shifted to the right.There then exists on the surface of the substrate, a tightly held,adsorbed system consisting of the new cation and the adsorbed waterhydroxyl. The major discovery of this invention is that surfaces somodified by cations are antimicrobial to any microorganism thatphysically contacts them.

The second critical aspect of this invention is the fact that themicroorganisms must physically contact the altered surface. By way ofexample, if the substrate is glass beads which have been treated asdiscussed above and the media is aqueous, for example, milk, and the twoare mixed together and stirred with a spatula for a few minutes, thenthis would be sufficient force to get the desired effect. Similarly,flowing a thin film of beer across an altered surface, such aspolyethylene, would be enough force to get the desired effect. A thirdexample would be stirring a solution containing the appropriatematerials with a powered stirrer with enough speed to make a vortex inthe solution. A fourth example would be blowing contaminated air througha filter, such as a furnace filter, which has been treated to make itssurface antimicrobial, according to this invention. The amount of forcerequired is a function of the particular system being used.

Once the surface of the substrate has been altered, microorganisms thatcontact the surface are destroyed. In certain examples there is notransfer of cationic material into the media and since the adsorbedsurface is not susceptible to erosion or leaching, the substrate can beused over and over without destroying its effectiveness.

Contemplated within the scope of this invention is any substrate whichdevelops a negative charge in water.

A surface suitably treated is essentially impervious to attack bymicroorganisms. A surface in a medium which supports the growth ofmicroorganisms has no apparent effect upon organisms in the medium, butorganisms that contact the surface are killed. If the medium is stirredto cause organisms to contact the surface, the organisms can all bekilled. In the absence of stirring, no noticeable number of organismsare killed, but the surface is protected from attack by the organisms,thus, there is a preservative effect contemplated within the scope ofthis invention.

Substrates that develop negative charges in water at a pH below about 10are suitable for the method of this invention. These include all knowntextile fibers, such as cotton, cellulose acetate, polyesters, nylon,wool, rayon, acrylon, etc.; all known organic surfaces, such as paints,polystyrene, silicone polymers, wood, rubber, etc.; almost all inorganicsurfaces, such as silica, sand, earth, silicate minerals, alumina,glass, including concrete and masonry surfaces etc. which are notsoluble in water.

Substances capable of making these substrates antimicrobial are thosethat ionize in water to form cations to exchange with protons in thelayer of water adsorbed onto the substrate. These include such diversematerials as sodium chloride, ammonium chloride, every type of amine,primary, secondary or tertiary, as well as quaternary ammonium salts,diamines, isothiuronium salts, sulfonium salts, and phosphonium salts.No antimicrobial activity of the substances in solution is required noris the activity in solution of any influence on the level ofantimicrobial activity shown by surfaces of substrates treated withthese substances.

Cations that contain bulky organic substituents are adsorbed to surfacesmore strongly. They do not give rise to more active surfaces, but theydo give rise to surfaces that are more resistant to loss of activitythrough loss of the cation from the surface.

Cations that contain silyl substituted organic substitutents have aunique advantage over other cations. Cations having substituents such as(CH₃ O)₃ Si- hydrolyze and polymerize and become essentiallyirreversibly bound.

The manner of putting these substances on the surface of a substrate iscritical to a degree. It is possible to spray, dip, paint or roll on thecationic material or in the case of particulate solids dispersions maybe used or in the case of fibers soaking techniques may be used. Thelength of time that contact between the cationic material and thesubstrate is necessary depends on the substrate and the type of cationicmaterial being used. Fibers for example should not be treated for longperiods of time unless the surface of the internal fibers is to betreated. Times of contact can range from a few seconds to a few hoursand the time required is that time necessary to exchange the hydrogenions of the adsorbed water on the surface for cations.

In actual practice, it is sometimes desirable to rinse the surface ofthe substrate after being treated.

The time required for rinsing the surface is not critical. Generallywater is used for this purpose but water and organic solvents can beused. For example, a rinse with a mixture of alcohol and water and thena clear rinse with water is generally sufficient.

Some of the advantages of the method of this invention are: Subtanceschosen for use in this invention may be inoccuous compounds that are nottoxic, corrosive, or hazardous to any appreciable extent. They do nothave to be biologically hazardous substances.

Exceedingly small quantities of the substances are capable of killing anapparently limitless number of organisms that contact a properly treatedsurface.

It can be noted, for example, that in Test No. 13 of Example 1, as lowas 0.03 g. ion/100 A² was sufficient to give 100% reduction ofmicroorganisms in only 1 treatment.

No substance needs to enter media in which the microorganisms are found.Thus a medium need not be contaminated by an antimicrobial agent toobtain a very strong antimicrobial effect.

A properly treated surface is capable of killing most diverse spectra ofmicroorganisms.

Now, so that those skilled in the art can better understand andappreciate this invention, the following examples are given. Theexamples following hereafter give those skilled in the art the guidancenecessary to practice this invention.

EXAMPLE 1

Many substrates were coated with materials by a "Paint on Technique". Bythis method a known amount of a chemical in solution was used to wet asolid. The solvent was evaporated and the amount of chemical in solutionwas deposited on the surface of the solid.

Table I shows surprising results obtained when finely divided powdersare mixed mechanically with cultures of microorganisms in what is called"The Powder Test".

In the Powder Test, a quantity between 0.1 and 0.5 g., depending uponthe particle size and density of a finely divided solid, was weighedinto a clean Pyrex test tube. A sample treated with a chemical by thePaint on Technique, and an equal sized sample of untreated sample wereeach mixed thoroughly (on a Vortex mixer) for 15 seconds and placed inan incubator for 30 minutes at 37° C.

Each sample was then agitated for 10 seconds with 10 ml. of sterilebroth. A 0.1 ml. portion of the broth was agar plated and incubated 18hours. After this time the number of viable organisms extracted from atreated sample was compared with the number extracted from an untreatedsample. The percent reduction of viable cells was calculated from thedifference between these values, i.e. (No. of cells from untreatedsample-No. of cells from treated sample divided by No. of cells fromuntreated sample)×100=% Reduction.

If no living organisms were extracted, the sample was said to haveachieved 100% kill.

This technique permits the evaluation of the antimicrobial activity of alarge surface area in relatively small volumes.

The data in Table I was obtained by use of 0.1 ml. of 1:100 dilution ofan 18 hour culture of Escherichia coli B.

In many cases the sample was washed and extracted with water beforebeing subjected to the powder test. Typically, about 1 g. of sample wasmixed thoroughly with about 10 ml. of distilled water and the mixturewas filtered or centrifuged and sucked dry in a vacuum filter. Sometimessamples were washed in this way many times.

In Table I, the following compositions are shown wherein the followingTest Nos. correspond to the Test Nos. in the Table:

    ______________________________________                                        Test   Substance                                                              No.    tested                                                                 ______________________________________                                        1      (CH.sub.3).sub.4 N.sup.+ Cl.sup.-                                      2      "                                                                      3      "                                                                      4      "                                                                      5      "                                                                      6      "                                                                      7      "                                                                      8      "                                                                      9      Na.sup.+ Cl.sup.-                                                      10     NH.sub.4.sup.+ Cl.sup.-                                                11     C.sub.18 H.sub.37 N(CH.sub.3).sub.3.sup.+ I.sup.-                      13     "                                                                      14     "                                                                      15     C.sub.18 H.sub.37 N(CH.sub.3).sub.2                                    16     Zephiran.sup.(j)                                                       17     n-C.sub.6 H.sub.13 NC.sub.5 H.sub.5.sup.+ Cl.sup.-                     18     "                                                                      19     n-C.sub.4 H.sub.9 N(CH.sub.3).sub.3.sup.+ Cl.sup.-                     20     (n-C.sub.4 H.sub.9).sub.2 N(CH.sub.3).sub.2.sup.+ Cl.sup.-             21     n-C.sub.4 H.sub.9 n-C.sub.8 H.sub.17 N(CH.sub.3).sub.2.sup.+                  Cl.sup.-                                                               22     (C.sub.2 H.sub.5).sub.3 N                                              23     "                                                                      24     n-C.sub.4 H.sub.9 NH.sub.2                                             25     "                                                                      26     (C.sub.2 H.sub.5).sub.2 NH                                             27     "                                                                      28     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.3.sup. +                Cl.sup.-                                                               29     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.3.sup.+                 Cl.sup.-                                                               30     "                                                                      31     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.3.sup.+                 OH.sup.-                                                               32     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(C.sub.2 H.sub.5).sub.3.sup.           + I.sup.-                                                              33     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(C.sub.2 H.sub.5).sub.3.sup.           + Cl.sup.-                                                             34     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(C.sub.2 H.sub.5).sub.2         35     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 NHCH.sub.2 CH.sub.2 NH.sub.2     36     {(CH.sub.3).sub.3 SiO}.sub.3 Si(CH.sub.2).sub.3 NHCH.sub.2                    CH.sub.2 NH.sub.2                                                      37     "                                                                      38     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 NC.sub.5 H.sub.5.sup.+                  Cl.sup.-                                                               39     "                                                                      40     "                                                                      41     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2 C.sub.8               H.sub.17.sup.+ Cl.sup.-                                                42     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2 C.sub.4               H.sub.9.sup.+ Cl.sup.-                                                 43     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2 C.sub.18              H.sub.37.sup.+ Cl.sup.-                                                44     "                                                                      45     "                                                                      46     "                                                                      47     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 P(n-C.sub.4 H.sub.9).sub.3.su           p.+ I.sup.-                                                            48     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 P(C.sub.6 H.sub.5).sub.3.sup.           + I.sup.-                                                              49     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 SC(NH.sub.2).sub.2.sup.+                Cl.sup.-                                                               50     CH.sub.3 SC(NH.sub.2).sub.2.sup.+ Cl.sup.-                             51     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 S(CH.sub.3)C.sub.18 H.sub.37.           sup.+ I.sup.-                                                          52     "                                                                      53     (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 S(CH.sub.3)C.sub.2 H.sub.5.su           p.+ I.sup.-                                                            54     {(CH.sub.3).sub.3 SiO}.sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2      55     O{Si(CH.sub.3).sub.2 (CH.sub.2).sub.3 NHCH.sub.2 CH.sub.2 NH.sub.2            }.sub.2                                                                56     {(CH.sub.3).sub.3 SiO}.sub.2 SiCH.sub.3 (CH.sub.2).sub.3 NHCH.sub.            2 CH.sub.2 NH.sub.2                                                    ______________________________________                                         .sup.(j) Zephiran® is a mixture of alkyl (40% C.sub.12, 50% C.sub.14,     10% C.sub.16) benzyldimethylammonium chlorides. (Winthrop Laboratories)  

                  TABLE I                                                         ______________________________________                                        Percent Reduction Of Viable Cells Of Escherichia coli-B By                    Surfaces Treated By The "Paint-on" Technique                                                       % Reduction                                              Test                      g. ion/                                                                              Un-                                          No.  Substrate   Solvent  100 A.sup.2                                                                          washed Washed                                ______________________________________                                        1    ®Minusil.sup.(a)                                                                      Methanol 10     100                                          2    "           "        8.2    90     85                                    3    "           "        0.2.sup.(b)   94                                    4    Cellulose   Water    540    30     77                                         Acetate.sup.(c)                                                          5    ®Ludox.sup.(d)                                                                        "               100    100                                   6    S/AN.sup.(e)                                                                              Water    8.2.sup.(b)   52                                    7    "           "        .14.sup.(b)   38                                    8    (CH.sub.3 SiO.sub.3 /.sub.2).sub.n.sup.(f)                                                "               100    100                                   9    (CH.sub.3 SiO.sub.3 /.sub.2).sub.n.sup.(g)                                                "        (g)    100    37                                    10   Minusil     "        10.5   62     51                                    11   Minusil     Ethanol  9.4           100                                   13   "           "        0.03.sup.(h)   100.sup.(h)                          14   S/AN        "        15.1   100    100                                   15   ®Cabosil.sup.(i)                                                                      Hexane   0.1    70                                           16   Minusil     Methanol 10     100    100                                   17   "           Water    10     100    24                                    18   S/AN        Water    10     44     18                                    19   Minusil     Methanol 10     99     90                                    20   "           "        10     100    100                                   21   "           "        10     100    93                                    22   "           "        10     53     38                                    23   S/AN        "        10     64     45                                    24   Minusil     "        10     23      0                                    25   S/AN        "        10     100    46                                    26   Minusil     Methanol 10     25     14                                    27   S/AN        "        10     24     17                                    28   Minusil     Water    10     99     90                                    29   Cellulose   "        10     95     100                                        Acetate                                                                  30   S/AN        "        10     84     30                                    31   Minusil     "        10     91                                           32   "           Methanol 10     87     43                                    33   "           "        10     69     33                                    34   "           "        10     100                                          35   "           Hexane   130    100    97                                    36   Cellulose   Water    10     100    100                                        Acetate                                                                  37   Minusil     Hexane   10     100    100                                   38   "           Methanol 10     100    100                                   39   "           Water    10     60     21                                    40   Ludox       "        10     100     100.sup.(k)                          41   S/AN        "        10     97     14                                    42   "           "        10     63     57                                    43   Minusil     Hexane   10     98     96                                    44   "           Methanol 10     100    100                                   45   "           Ethanol  1      75                                           46   S/AN        Water    10     100    100                                   47   Minusil     "        100    100    23                                    48   "           "        100    100    20                                    49   "           "        10     100    96                                    50   "           "        10     100    91                                    51   "           "        10     96     83                                    52   S/AN        "        10     100                                          53   Minusil     "        10     100    65                                    54   "           Hexane   10     100    48                                    55   "           "        164    100    100                                   56   "           "        170    100    100                                   ______________________________________                                         .sup.(a) 99.9.sup.+ % silica, 10μ particle size, 1.1 m.sup.2 /g surfac     area (Pennsylvania Glass Sand, Co.)                                           .sup.(b) This sample was prepared with radioactive (CH.sub.3).sub.3.sup.1     CH.sub.3 N.sup.+ Cl by Substantive Method.                                    .sup.(c) Cellulose Acetate fiber, 10 denier 0.1 m.sup.2 /g surface area       (Celanese Corp.)                                                              .sup.(d) LudoxHS, 30% silica sol in water, pH 9.8, 210 m.sup.2 /g surface     area 15 mμ particle size.                                                  .sup.(e) S/AN, StyreneAcrylonitrile microcapsules, 1 to 20μ particle       size, 0.63 m.sup.2 /g surface area (Dow Chemical Co.)                         .sup.(f) Methyltrimethoxysilane (13.7 g., 0.1 mole) and                       tetramethylammonium chloride (1.2 g., 0.015 mole) was dissolved in 5 ml.      of water and methanol and water was distilled from the solution as a          polymeric gel formed. The gel was dried to an insoluble white solid which     was ground to a fine powder. The powder was washed exhaustively with wate     until the wash water contained no detectable chloride ion by test with        silver nitrate.                                                               .sup.(g) Same as (f) with sodium chloride. Unwashed powder contained 2.6%     Na.sup.+, 0.79% Cl.sup.-, washed powder contained 0.07% Na.sup.+, nil         Cl.sup.-.                                                                     .sup.(h) Prepared with C.sub.18 H.sub.37 N(CH.sub.3).sub.2.sup.14             CH.sub.3.sup.+ I.sup.- applied at a low level and washed with water,          ethanol and dilute hydrochloric acid. Concentration of the radioactive        cation on the solid was measured by radioactive assay.                        .sup.(i) Cabosil MS75, silica aerogel, 12-15 mμ particle size, 274         m.sup.2 /g (Cabot Corp).                                                      .sup.(k) Washed exhaustively with water until free of detectable chloride     ion.                                                                     

The data shown in Table I indicates a number of unexpected effects. Mostsurprising is the fact that every substance tested by the Powder Testdisplayed antimicrobial behavior. This was most surprising since many ofthe substances have little or no antimicrobial activity in solution.

The most widely standardized method of measuring antimicrobial activityof any substance is probably that of Serial Tube Dilution. In thistechnique, 1 g. of substance is mixed with 9 ml. of sterile broth in asterile glass tube. One ml. of this mixture is transferred asepticallyto a second tube containing 9 ml. of broth to create a 1/100 dilution ofthe substance being tested. This is repeated to form a series ofdilutions each increasing by a factor of 10. Thus 1/10, 1/100, 1/1000,1/10,000 etc. The broth is selected to contain vitamins, amino acids,sugars, etc. selected for optimum growth of the microorganism to be usedfor the test.

Each of the series of tubes is inoculated with about 0.1 ml. of a testorganism and incubated for 24 hours at 37° C. for bacterial growth or at25° C. for fungal growth. After incubation the most dilute concentrationof substance to inhibit growth of the organism is recorded as theMinimum Inhibitory Concentration (MIC). This is usually expressed asparts per million (PPM) or μg/ml., i.e. in parts by weight of broth.

In most of the tests of Table I, the amount of the substance tested inthe Powder-test was insufficient to kill the E. coli-B by dissolvingfrom the substrates to enter the culture. Calculations of the μg ofsubstances on the unwashed samples, if completely dissolved in 0.1 ml.of culture, indicate that such solution could not equal the MIC of thesubstance. The amount of substance after washing is unknown, but it wasnecessarily less.

Table II indicates the MIC, for E. coli-B for examples for which thisvalue is known and lists the maximum concentration of the substancesthat could be reached if all the substances were dissolved from thesurfaces of the substrates. The test nos. refer to Table I.

                  TABLE II                                                        ______________________________________                                                                             Max.                                     Test                        MIC      μg/ml.                                No.  Substance              μg/ml in test                                  ______________________________________                                         3   (CH.sub.3).sub.4 N.sup.+ Cl.sup.-                                                                    >10,000  <175                                     10   NH.sub.4.sup.+ Cl.sup.-                                                                              >10,000  <250                                     13   C.sub.18 H.sub.37 N(CH.sub.3).sub.3.sup.+ I.sup.-                                                    100      <3                                       19   n-C.sub.4 H.sub.9 N(CH.sub.3).sub.3.sup.+ Cl.sup.-                                                   >10,000  <2700                                    28   (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.3.sup.+                 Cl.sup.-               >10,000  <300                                     38   (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 NC.sub.5 H.sub.5.sup.+                  Cl.sup.-               >10,000  <3200                                    42   (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2 n-C.sub.4             H.sub.9.sup.+ Cl.sup.- >10,000  <2000                                    41   (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2 C.sub.8               H.sub.17.sup.+ Cl.sup.-                                                                              >10,000  <2600                                    45   (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2 C.sub.18              H.sub.37.sup.+ Cl.sup. -                                                                             1,000    <200                                     ______________________________________                                    

The examples of Table I indicated that every substance that dissolved inwater and ionized in water to form cations other than protons werecapable of making surfaces antimicrobial as measured by the Powder-Test.Every salt was effective and every amine. Amines are well known todissolve in water to form ammonium hydroxides, e.g. R₃ N+H₂ O⃡R₃ NH⁺+OH⁻.

The fact that certain substrates that had been treated with chloridesalts and were washed until free or almost free of chloride ion,indicated that the cation from each substance was the essentialactivating species that caused surfaces to exhibit antimicrobialbehavior.

This was most surprising and also most evident with examples such as No.9, Na⁺ Cl⁻ ; No. 7, (CH₃)₄ N⁺ Cl⁻ ; No. 13, C₁₈ H₃₇ N(CH₃)₃ ⁺ I⁻ ; orNo. 40, (CH₃ O)₃ Si(CH₂)₃ NC₅ H₅ ⁺ Cl⁻. The salt of No. 40 has an N/Clratio of unity, but elemental analysis of the washed Ludox indicated anN/Cl ratio of about 296/1. Although essentially all of the chloride ionwas not on the substrate, the surface was highly anti-microbial.

EXAMPLE 2

To gain an analytical capability to show what occurred on a surface thatcaused it to become antimicrobial, typical substances were maderadioactive. Radio-tracer techniques were then applied to measureaccurately very low concentrations of the substances and to locate thesubstances. (A) (CH₃)₃ N¹⁴ CH₃ ⁺ Cl⁻ was prepared by sealing ¹⁴C-methylchloride (1.96 mg., 0.039 m mole), (0.252 m Ci) withmethylchloride (218.3 mg., 4.5 m mole) in methanol (0.83 g) andtrimethylamine (1.8118 g., 30.71 m mole) in a flask at room temperature.Crystals formed until the mixture solidified. Vacuum was applied toremove all volatile materials leaving 2.9616 g. of fine white crystalsof radioactive tetramethylammonium chloride with an activity of 0.025mCi/g. (B) C₁₈ H₃₇ N(CH₃)₂ ¹⁴ CH₃ ⁺ I⁻ was made in essentially the sameway from C₁₈ H₃₇ N(CH₃)₂, CH₃ I and ¹⁴ CH₃ I in methanol. The productwas recrystallized from ethanol to obtain 520.9 mg. with an activity of3.28 mCi/g. (C) (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₂ C₁₇ H₃₅ ⁺ Cl⁻ was madefrom octadecyl-1-¹⁴ C chloride in hexane with (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂in methanol in a sealed glass tube at 110° for 70 hours. The product waswashed with hexane and dried to give 10 mg. of crystals with an activityof 1 mCi/m mole. The hexane was used to dissolve and recrystallize 967.7mg. of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ to obtain 1.023 g. ofradioactive crystals with a specific activity of 0.36 mCi/m mole. (D)(CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₃ ⁺ Cl⁻ was prepared from3-dimethylaminopropyltrimethoxysilane (2.44 g., 12 m moles) in methanol(1.0 g) and ¹⁴ CH₃ Cl (3.89 mg., 0.5 mCi) at 100° C. for 2 hrs in asealed ampoule. All volatile materials were removed from the productunder vacuum leaving white crystals (2.98 g., 98% yield).

Solutions of (B), (C), (D) were prepared in distilled water, each 10⁻³ Mconcentrations. Each of these solutions was stirred with sufficientMinusil so that if the solutions were depleted of (B), (C) or (D), theMinusil should have 10 molecules of radio-cations per 100 A² of thesurface of the Minusil. The stirring was for the time and at thetemperatures indicated in Table III. The Minusil was removed from thesolutions, washed repeatedly with water and assayed.

                  TABLE III                                                       ______________________________________                                        Rate and Level of Adsorption on Minusil                                       Substance ° C.                                                                             Hours       g-ion/100 A.sup.2                             ______________________________________                                        (B)       25        2           1                                                       25        24          1                                                       60        2           1                                                       60        24          1                                             (C)       25        2           3.8                                                     25        24          4.8                                                     25        168         7.2                                                     25        336         7.4                                                     60        2           6.5                                                     60        4           7.8                                                     60        24          9.62                                          (D)       25        Seconds     .7                                                      25        Seconds     .13.sup.(a)                                   ______________________________________                                         .sup.(a) Cellulose Acetate fibers, 0.12 m.sup.2 /g surface area.         

These data indicate that the adsorption of (B) was limited to onemolecule/100 A², but that (C) was adsorbed without limit until thesolution was depleted.

This method in which the cations are adsorbed by a surface from a verydilute solution is referred to as the Substantive Method. This method isnot like that of the Paint-on Technique by which all of a substance iscoated onto or mixed with surfaces. Both methods produce activesurfaces, but the surfaces are not necessarily the same.

By the Paint-on Technique (D) was put onto Minusil and Cellulose Acetatefiber and the effect of water washing, level of treatment and the effectof drying the solids before they were washed was studied.

                  TABLE IV                                                        ______________________________________                                        (D) (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2.sup.14            CH.sub.3.sup.+ Cl.sup.- on Minusil                                            Temp. of   g-ion/100 A.sup.2                                                  Drying °C.                                                                        initially   1 wash     2 wash                                      ______________________________________                                        ˜25  2.3         1          1                                           ˜25  9.5         1.8        1                                            70        9.3         1.4        1                                            100       4.6         1.1                                                    ˜25  4.8         1.1                                                    ˜25  25.5        2.4        1.3                                         ˜25  20.sup.a    .9.sup.a                                               ______________________________________                                         .sup.a Cellulose Acetate                                                 

The Substantive Method was employed at room temperature by stirringpowders for 2 hours with enough (C) to apply 10 molecules/100 A². After2 hours, the powders were separated in a centrifuge. They were assayedand then water washed 3 times in a centrifuge and assayed again.

                  TABLE V                                                         ______________________________________                                        Substantive Treatment With (C) At Room Temperature                                       g. ion/100 A.sup.2 of surface                                      Substrate    As Prepared    Washed                                            ______________________________________                                        Minusil      3.8            3.7                                               S/AN         7.6            6.9                                               Teflon.sup.a 2.5            2.3                                               ______________________________________                                         .sup.a Finely powdered Teflon, 0.012 m.sup.2 /g surface area.            

These data indicate that such diverse surfaces as silica,polystyrene-acrylonitrile, cellulose acetate and Teflon adsorb cations.The rate at which the ions are adsorbed is very fast for the firstmolecular layer of ions. Compounds (B) and (D) are adsorbed verystrongly up to approximately a limit of one molecular layer. Compound Cis adsorbed slowly after about the first molecular layer, but it isadsorbed without limit until the solution is depleted. If excess (B) and(D) is applied by the paint-on technique, excess is removable by wateruntil the level near one molecular level remains.

Every sample in Tables III, IV aind V have near 100% reduction of E.coli-B in the powder test.

Fibers of Cellulose Acetate, Polyester and Nylon-66 (0.47 to 0.49 g)were stirred in 100 ml. of 0.001% (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₂ C₁₇H₃₅ ⁺ Cl⁻ in water at room temperature. Periodically 0.1 ml. of thesolution was withdrawn and assayed in a liquid scintillationspectrometer to measure the rate at which the solution was depleted ofradioactive cations. After 24 hours the fibers were washed thoroughlywith distilled water and assayed.

                  TABLE VI                                                        ______________________________________                                        Substantive Treatment of Fibers with                                          (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2.sup.14 CH.sub.2       C.sub.17 H.sub.35.sup.+ Cl.sup.-                                              Decompositions/min/g of Solution                                              Cellulose Acetate       Polyester                                                                              Nylon-66                                     Minutes pH 5     pH 7.5   pH 10  pH 7.5 pH 7.5                                ______________________________________                                        0.0     159      163      167    169    167                                   0.5     121      125      114                                                 5       118      116      102    126    102                                   15      100      107       97    102    104                                   30       94       99       93     81     94                                   1440     52       56       71            89                                   Count on                                                                      Washed                                                                        Samples                                                                       dec/min/g                                                                             14,581   16,317   20,808 18,483 14,719                                Cations/100                                                                   A.sup.2 of                                                                    Surface 1.3      1.4      1.8    1.6    0.8                                   ______________________________________                                    

Cellulose acetate fibers in a 1% solution as above adsorbed 210cations/100 A² of surface.

Fibers (10 g.) of Cellulose Acetate, Polyester, Cotton and Fiberglasswere stirred in 100 ml. of 0.4% (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₂ C₁₇ H₃₅⁺ Cl⁻ in water at room temperature. The stirring was for a short timeuntil they were thoroughly wetted by the solution. They were then takenfrom the solution, pressed as dry as possible on filter paper andassayed for radioactivity.

The samples were then washed with distilled water for five hours with achange of water every 10 minutes for the first hour and every hour forfour hours.

                  TABLE VII                                                       ______________________________________                                                   Initial Activity                                                                             After Washing                                       Fiber      mCi/g          mCi/g                                               ______________________________________                                        Polyester  .49            .15                                                 Cotton     .82            .69                                                 Cellulose                                                                     Acetate    .45            .44                                                 Fiberglass .50            .16                                                 ______________________________________                                    

The polyester fiber (0.5 g) was soaked in 0.01% concentration of abovesolution for 24 hrs. and assayed. The fiber had adsorbed 3 cations/100A² of surface.

A second (0.5 g) sample was soaked in the same solution for 24 hours.This sample adsorbed 2 cations/100 A² of surface.

A third sample (0.5 g) was soaked in the same solution 74 hours. Thissample adsorbed 5 cations/100 A² of surface.

A fourth sample (0.5 g) was soaked in the same solution 24 hours. Thissample adsorbed less than 1 cation/100 A² of surface.

The solution was then assayed and had no detectable radioactivity,indicating that the solution had been depleted of all radio cations.

EXAMPLE 3

Minusil was treated with C₁₈ H₃₇ N(CH₃)₃ ⁺³⁶ Cl⁻ as nearly identicallyas possible to the sample in Table III. A solution in water thatcontained 21.28×10⁻⁶ moles of chloride at a specific activity of 77.823dpm/μmol of Cl⁻ was used to treat 1.0073 g. of 10μ Minusil.

Unwashed powder indicated only 0.16 Cl⁻ per 100 A² of surface. One washwith 15 ml. of water reduced the Cl⁻ level to zero within the limits ofdetection of ³⁶ Cl⁻, which we estimate to be about 0.005 Cl⁻ /100 A².

The activity of the water remained unchanged during this experimentindicating all of the chloride ion remained in the water and none becameadsorbed onto the Minusil although 1 cation/100 A² of surface wasadsorbed.

This is taken as compelling evidence that the cation was adsorbed ontothe surface by a process of ion exchange, most likely described by anequation such as ##STR5## In this equation, M⁺ is a cation other thanH⁺, and an H⁺ is displaced from water adsorbed onto the substrate tobecome hydrated and to enter the aqueous phase as H⁺.sub.(aq).

Our data indicates that this equation is quantitatively accurate up tothe point that the concentration of M⁺ near the surface of the substratereaches a limit of about one M⁺ ion/100 A² of surface area.

A sample of Minusil with one non-radioactive Me₃ NC₁₈ H₃₇ ⁺ cation per100 A² was stirred in a dilute aqueous solution that contained a largeexcess of C₁₈ H₃₇ N(CH₃)₂ ¹⁴ CH₃ ⁺ cations. After two hours at roomtemperature, the Minusil assayed as having 1.0 radioactive cations/100A² of surface.

This indicated that the process of ion exchange took place betweenradioactive and non-radioactive cations on the surface and that anequilibrium was established rapidly in this example. Thus:

    Surface·OH.sup.- ·M.sup.,+.sub.(aq) +M.sup.+ ⃡

    Surface·OH.sup.- ·M.sup.+ +M.sup.,+.sub.(aq).

This further indicates that simple cations do not adsorb beyond onecation per 100 A² by the Substantive Method from dilute solutions.

A sample of Minusil with one non-radioactive Me₃ NC₁₈ H₃₇ ⁺ cation per100 A² was stirred with a dilute aqueous solution containing a largeexcess of (CH₃)₃ N¹⁴ CH₃ ⁺ Cl⁻ as above. Subsequent assay showed noactivity on the surface.

This indicated that a small cation such as (CH₃)₄ N⁺ cannot measurablydisplace a large one according to the above equation.

This agrees with commonly accepted theory that cations that associate insolution, e.g., form micelles at suitable concentrations, adsorb tosolids much more strongly than do smaller cations.

EXAMPLE 4

The experiments of Example 3 which used (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₂C₁₇ H₃₅ ⁺ Cl⁻ were repeated with (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₅ ⁺³⁶Cl⁻.

Minusil in Table III which had adsorbed 3.8 cations/100 A² was assayedfor ³⁶ Cl⁻ and 1.89 ³⁶ Cl⁻ ions/100 A² was found. The sample was washedfurther and the ³⁶ Cl⁻ was reduced to 1.1 ³⁶ Cl⁻ ions/100 A².

Minusil (1 g.) was stirred at 60° C. for 24 hours with 2×10⁻³ Molarsolutions (100 ml) of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₂ C₁₇ H₃₅ ⁺ Cl⁻ andof (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺³⁶ Cl⁻. Assay of these samplesshowed 16.3 cations and 4.3 anions per 100 A² of surface. Exhaustivewater washing of these samples had very little effect upon these values.Washing with ethanol reduced them to 13.4 cations and 1.0 anions/100 A².

A mixture of 92 mg (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₂ C₁₇ H₃₅ ⁺ Cl⁻ and2.1851 g. (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ was hydrolyzed withwater and freed of all volatiles under vacuum at room temperature orbelow to obtain a powder essentially (HO)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺Cl⁻ with a specific activity of 1.4719×10⁸ dpm/g.

The experiment was repeated with 3.1 g. of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈H₃₇ ⁺³⁶ Cl⁻.

Solutions of these salts, 7×10⁻⁴ Molar and 0.40 g. of Minusil werestirred 2 hours at room temperature and water washed. The Minusiladsorbed 3.5 cations/100 A², nearly duplicating the value in Table IIIunder nearly the same conditions. The Minusil had adsorbed no detectablelevel of ³⁶ Cl⁻.

These data indicate that the adsorption of the silyl substituted saltdiffered from the adsorption of a simple ammonium salt, by not beinglimited to one cation/100 A² of surface. Adsorption beyond this levelincluded some part which was adsorbed as an ion pair. If adsorption wasfrom sufficiently dilute solution, or if the surface was subjected tosufficient washing, most or all of the anions were absent from thesurface.

Precipitation of the polymeric hydrolyzate of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂C₁₈ H₃₇ ⁺ Cl⁻ from dilute solution in water in the absence of asubstrate occurs slowly. The precipitate was filtered from the water,dried and analyzed for chloride ion. The level was too low to measure bynon-radiotracer techniques. The dried precipitate was ground to a powderand gave 100% reduction of E. coli-B in the powder test. The precipitatewhen dried at 100° C. is polymeric and no longer soluble in water.

These data indicate that the silyl substituted salts can polymerize inthe absence of a substrate to form an insoluble product which is formedby a process in which ion exchange was only one part. The polymer was apolysiloxane containing polymer units approximately described as HO⁻ O₃/₂ Si(CH₂)₃ N⁺ (CH₃)₂ C₁₈ H₃₇ Cl⁻.

EXAMPLE 5

A sample (10 mg., 100 cm²) of Minusil having 3.8 (CH₃ O)₃ Si(CH₂)₃N(CH₃)₂ ¹⁴ CH₂ C₁₇ H₃₅ ⁺ cations per 100 cm² of surface was added to 2ml of resting state E. coli-B culture containing 3750 cells/ml in asmall closed vial, Vial-1.

A sample (10 mg) of untreated Minusil was added to 2 ml of the cells inVial-2.

Only the culture was added to Vial-3.

The three vials were fastened to a wheel at a 45° angle and tipped endfor end 24 times/min. as the wheel rotated in an incubator at 37° C.Samples were removed from the vials periodically and counts were made ofthe viable cells/ml. at various times. The results are shown in TableVIII.

                  TABLE VIII                                                      ______________________________________                                        Viable Cells/ml E. coli-B                                                     Minutes   Vial-3      Vial-2     Vial-1                                       ______________________________________                                         0        3750        3750       3750                                          5        4370        3900       1600                                         15        5000        3700        29                                          30        4200        4300         1                                          45        4100                     3                                          60                    4400         0                                          ______________________________________                                    

Vials 3 and 2 showed an increase in the number of cells. Vial-1contained no more than about 4 μg of active substance per ml. ofculture, or less than 4% of the (MIC) for the substance. Under theconditions of this test, this small amount caused rapid reduction in thenumber of viable organisms in the culture.

A plot of the logarithm of the number of viable cells versus time forthe cells in Vial-1 gave a straight line. See FIG. 1, which is a plot ofthe number of viable cells on a log₁₀ scale versus minutes on a linearscale.

This plot indicates that the organisms were killed by physical contactwith the treated surface and that the rate of decrease in the numberthat were viable under standardized conditions of agitation followed amathematical equation very like one for the equation for the kinetics ofa second order reaction. Thus the rate of decrease is: ##EQU1## in whichS=Concentration of viable organisms/ml

K=A specific rate constant

t=time

During an experiment the area remains constant. Integration thereforegives:

    -Ln S=K{Area/ml}t+constant

a plot of Ln S versus t called for by this equation is a straightlinewith a slope=K{area/ml}.

A convenient way to determine K is from the equation

    Ln S.sub.t /S.sub.o =K{area/ml}(t.sub.o +t)

where S_(o) is the concentration of organisms at t_(o) and S_(t) is theconcentration of organisms at time, t.

The time required to kill half of the organisms can be t₁ /₂ and

    Ln 0.5=0.693=K{area/ml}t.sub.1 /.sub.2

From FIG. 1, t₁ /₂ is about 3.25 minutes and area/ml=50 cm² /ml. ThenK=0.004 ml/cm² /min.

This calculation says that the treated surface under these conditions ofagitation killed half of the organisms every 3.25 minutes and itcontinued to do this until the viable organisms were very few.Experimentally, viable organisms vanished in about 1/2 hr. althoughmathematically this should require an infinite amount of time.

Experiments were repeated with a range of concentrations of cells, S_(o)and surface areas/ml, see Table IX.

                  TABLE IX                                                        ______________________________________                                              Viable Cells of                                                               E. coli-B-   Minusil-                                                   cm.sup.2 /ml                                                                        S.sub.o      t.sub.1/2   K(ml/cm.sup.2 /min)                            ______________________________________                                        25    10,270       5.16        0.005                                          50    4,639,000    3.2         0.004                                                             3.0.sup.(a) 0.005.sup.(a)                                                     7           0.002.sup.(b)                                        8,243        3.6         0.004                                                3,750        3.3         0.004                                                1,459        1.7         0.008                                                992          3.3.sup.(a) 0.004.sup.(a)                                  100   7,502        1.75        0.004                                                1,314        1.68        0.004                                          ______________________________________                                         .sup.(a) surface of S/AN                                                      .sup.(b) substance (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3                      N(CH.sub.3).sub.3.sup.+ Cl                                               

The samples of Minusil and S/AN shown in Table V were studied to seewhat effect the level of treatment of a surface has upon the specificrate constant K. The results are shown in Table X.

(CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ (25 g.) was dissolved in 34.5 g.of methanol. Distilled water (15 g.) was added to the solution which wasthen heated to reflux and then all volatiles were removed under vacuumleaving a solid polymer, essentially {O₃ /₂ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺Cl⁻ }_(n). This solid was ground to a fine powder and washed with water.It was essentially insoluble in water. It was washed with ethanol andisopropanol and lost less than 2% by weight during these washings. Thewashed powder was dried and ground again and sieved. Particles smallerthan 150 μm were tested. The area/g of this powder was not accuratelyknown, so K for this powder could only be approximated as the 10 mg. ofthis powder had an area equal to 10 mg. of Minusil.

                  TABLE X                                                         ______________________________________                                        molecules/               K(ml/cm.sup.2 /min)                                  Substance                                                                             100 A.sup.2                                                                              Substrate E.coli-B                                                                             P Aeruginosa                              ______________________________________                                        C.sup.(a)                                                                             1          Minusil   0.002  0.003                                                        S/AN      0.001  0.001                                             7          Minusil   0.006  0.001                                                        S/AN      0.004  0.005                                             not applicable                                                                           none             0.008                                     B.sup.(b)                                                                             1          Minusil   0.008  0.002                                                        S/AN      0.003  0.001                                             7          Minusil   0.004                                                               S/AN      0.002                                            ______________________________________                                         .sup.(a) C is (CH.sub.3 O).sub.3 Si(CH.sub.2).sub.3 N(CH.sub.3).sub.2         C.sub.18 H.sub.37.sup.+ Cl.sup.                                               .sup.(b) B is C.sub.18 H.sub.37 N(CH.sub.3).sub.3.sup.+ Cl               

These data in Tables IX and X indicate that the surfaces have remarkablysimilar effects upon organisms. Multiple layers of the organic cationmay slightly reduce the activity of a surface. Data of Table IIIsuggests that layers of the organic cation would be removed during thetest until only one layer remained. Thus the activity measured in thismanner for a simple cation, most probably is that of an adsorbed levelone cation thick on the substrate.

Multiple layers of the silyl-substituted cation seem to slightlyincrease the activity of the surface. The solid polymeric substance hadan activity similar to that of a substrate with numerous layers ofadsorbed cations. This suggests that as the number of layers of thesecations increase, the effects of the substrate vanish, so that asubstrate coated with multiple layers of a substance and the polymericsubstance alone present very similar effects upon contact withorganisms.

EXAMPLE 6

Nylon fibers were treated with sufficient dilute solution of (CH₃ O)₃Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ in water to deposit 0.1% by weight uponthe fiber if the solution were depleted. The fibers were washed withwater and submitted to the test described in Example 5.

This time 3 ml. of a tryptic soy broth was inoculated with 1 ml. of an18 hour culture of Staphylococcus aureus FDA-209 to give 4 ml. of liquidin the vials which were rotated 29 times/minute and approximately 10 mg.of fiber was tested. The surface area per gram of this fiber was notknown, but it was much less than that of the samples in Example 5.

                  TABLE XI                                                        ______________________________________                                        Viable cells of S. aureus in Tryptic soy broth                                Minutes  Vial-3      Vial-2      Vial-1                                       ______________________________________                                         0       19,000      19,000      19,000                                       30       38,000      20,000      16,000                                       60                   25,000      11,000                                       90       45,000                  7,000                                        120      100,000                 4,600                                        150      150,000     60,000      1,200                                        180                  90,000        700                                        ______________________________________                                    

The logarithm of the number of viable cells in Vial-1 plotted versustime was a slightly concave curve. This is due to the fact that in thenutrient medium, cells were multiplying during this test, so that thenet rate of their decrease was the difference between the rates at whichthey were dying and at which they were multiplying.

The experiment was repeated with 3 ml. of sterile brine replacing thesoy broth.

                  TABLE XII                                                       ______________________________________                                        Viable cells of S. aureus in Sterile Brine                                    Minutes     Vial-2        Vial-1                                              ______________________________________                                         0          15,000        15,000                                              30          4,000         880                                                 60          2,000          15                                                 90          1,500          2                                                  120         1,500          0                                                  ______________________________________                                    

The logarithm of the number of viable cells in Vial-1 in this caseplotted versus time was a straight line as in FIG. 1.

EXAMPLE 7

A 1/4"×7" piece of polyethylene tubing was filled with 6" of fine sand,6.920 g., (0.012 m² /g, total area 0.083 m²). A solution of 0.00538 g.,sufficient for 8 molecules/100 A² of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₂C₁₈ H₃₇ ⁺ Cl⁻ in 10 ml. of water was permitted to drip onto the columnof sand during 15 minutes, followed by 50 ml. of distilled water. Thecolumn of sand was then assayed. It was uniformly treated to a level of0.52 to 0.56 molecules/100 A².

The experiment was repeated with 5.218 g. of soil, which was mostly sandgently swirled in 3 ml. of the solution for 1/2 hour. It then had anactivity of 0.86 mCi/g.

Untreated soil (4.084 g.) was put into the polyethylene tube and treatedsoil (0.9757 g.) was put on top of it. Water was then trickled thru thecolumn very slowly, 275 ml. in 120 hrs. The water was assayed andcontained less than 0.8% of the radioactivity introduced as treatedsoil. The column of soil was assayed. All the radioactivity had remainedin the top portion of treated soil. No radioactivity could be detectedin the untreated portion of the soil, indicating that the activesubstance had not migrated during the long elution with water.

The treated soil both before and after it had been washed was tested bythe Powder Test with Staphylococcus faecalis. In both cases a 50%reduction in viable cells was obtained over that recovered from a sampleof the untreated soil.

Fine sand (2 g.) was sterilized with steam and wet with a 1% solution of(CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl. Sample 1 was sucked dry on afilter. Sample 2 was heated to 70° until it was dry.

These samples were tested by the Powder Test with 500,000 cells ofStaphylococcus faecalis, and compared with untreated sterilized sand.Recovered viable cells from Sample 1 were 5.6% and from Sample 2, 5.9%of the number recovered from untreated sand.

Diatomaceous earth (Celite-281) was treated in the same way. The Celitewas divided into three samples. Sample 1 was not washed. Sample 2 waswashed with 100 ml. of distilled water. Sample 3 was washed five timeswith 100 ml. of water. These samples were tested by the Powder Test with500,000 cells of E. coli-B or of S. faecalis and retested with 2,500,000cells of each.

Untreated Celite permitted recovery of viable cells too numerous tocount. Samples 1, 2 and 3 gave no recovery of viable cells.

Sand and Celite are both silica. The superior activity of treated Celitewas due to its having much more surface area per gram than the finesand.

The Celite (0.11 g.) was stirred with 2 ml. of 1% (CH₃ O)₃ Si(CH₂)₃N(CH₃)₂ ¹⁴ CH₂ C₁₈ H₃₇ ⁺ Cl⁻ in water and allowed to dry at roomtemperature. It then assayed as 69 molecules/100 A² of the radioactivecation. It was washed with 15 ml. of distilled water 3 times and thenassayed as 10 molecules/100 A².

Celite treated with only enough substance to put 1.8, 2.5, 2.8, 2.5molecules/100 A² was washed as above and assayed as having 1.7, 2.3,2.6, 2.5 molecules/100 A².

EXAMPLE 8

The Celite from Example 7 having 2.5 cations/100 A² (1.5% by weight) wasmilled into a natural rubber, heavy duty tire tread stock and cured at300° F. for 30 minutes to prepare Sample 1.

The Celite (1.5% by weight) was also milled into a passengercar-sidewall synthetic (SBR)-rubber tire stock and cured at 300° F. for30 minutes to prepare Sample 2.

Samples of these cured rubbers with and without the Celite were washedthoroughly with water and placed into petri dishes containing trypticsoy broth. Each dish was then sprayed with a mixture of fungal spores ofA. niger, A. flavus, A. versicolor, C. globosum and P. funiculosum andincubated at 25° C. for one week. At the end of one week visualinspection of the samples showed that fungi had grown extensively uponthe control samples that contained no treated Celite. Sample 1 had fungiupon about 7% of its surface. Sample 2 had fungi growing upon about 2%of its surface.

EXAMPLE 9

A very thin film of Cellulose Acetate was dipped into a 2% solution of(CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ and hung up to dry. 1"×1/2"pieces of the film were submerged in cultures of E. coli-B and S.Faecalis which each contained 1,000,000 cells/ml. They were removed andthen warmed to 37° C. for 30 minutes and submerged into tubes of sterilebroth and warmed to 37° C. for 18 hrs.

No viable organisms were detected in the broth after this period ofincubation with the treated film. Untreated film gave rise to growth oforganisms in the broth. The treated films had killed every organism theyhad encountered when they were submerged in the cultures.

EXAMPLE 10

Six inch squares of thin Silastic® sheet, Medical Grade Silicone Rubber,were swirled about in 100 ml. of toluene for 1.5 hours at roomtemperature. The toluene contained 0.2% by weight (A) (CH₃ O)₃ Si(CH₂)₃N(CH₃)₂ ¹⁴ CH₂ C₁₇ H₃₅ ⁺ Cl⁻ to give Sample 1. The toluene contained0.1% by weight (B) (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ ¹⁴ CH₃ ⁺ Cl⁻ to give Sample2.

The samples were removed from the toluene and hung up to dry at roomtemperature. One half of each sample was swirled about in 100 ml. ofdistilled water for 21/2 minutes. This was repeated 3 times with thefresh water each time. The washed half of Sample 1 became Sample 3. Thewashed half of Sample 2 became Sample 4. These samples were assayed forthe radioactive cations. Sample 1 assayed as 0.4 mg(A)/g. of Silastic®Rubber. Sample 3 assayed as 0.4 mg(A)/g. of Silastic® Rubber. Sample 2assayed as 0.5 mg(B)/g. of Silastic® Rubber. Sample 4 assayed as 0.4mg(B)/g. of Silastic® Rubber.

Six inch squares of the Silastic® Rubber sheet were swirled about in a2% by weight solution of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ inwater for a few minutes, removed from the water and dried on sterilefilter paper in sterile petri dishes. Half of each square was washedwith water as described immediately above.

A piece of each of the samples were placed in 20 ml. of sterile nutrientsolution or in tryptic soy broth. Each example was then sprayed with amixture of spores of A. niger, A. flavus, A. versicolor, C. globosum,and P. funiculosum and incubated for one week at 25° C.

At the end of the week the samples were examined visually. UntreatedSilastic® Rubber was overgrown completely with fungi. Unwashed sampleshad no fungi growing on them. Washed samples had a few spots on them,about 5% of the surfaces, upon which fungi were seen.

With these examples fungi flourished microscopically close to thetreated Silastic® Rubber but did not grow on the surfaces.

EXAMPLE 11

A typical latex paint was prepared with 100 g. of Rhoplex® AC35X latexpaint and 135 g. of a pigment slip. Paper squares were dipped into thepaint and hung up to dry. (Sample A).

Two ml. of a 50% solution of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ inmethanol was stirred into the paint and paper squares were dipped intothe paint and hung up to dry (Sample B).

These paper squares were then placed on the surface of malt agar inpetri dishes and inoculated with spores of Pullularia pullulans ATC-9348and incubated one month at 28°-30° C.

At the end of this time the fungus had grown over much of the squares ofSample A. Fungus was found on 2% of the surfaces of Sample B.

EXAMPLE 12

Square pieces of Ponderosa pine wood, (1.5"×1.5"×0.25") were soaked forfive minutes in 5% by weight solutions of (A) (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂C₁₈ H₃₇ ⁺ Cl⁻ and (B) (CH₃ O)₃ Si(CH₂)₃ NHCH₂ CH₂ NH₂.

Untreated pieces and pieces treated with (A) and (B) were placed onsterile Sabouraud agar and sprayed with a mixture of spores of A. niger,A. flavus, A. versicolor, C. globosum, and P. funiculosum and incubatedat 27° for 21 days. Untreated pine was completely overgrown with fungiin three days. Pine treated with (A) had very little fungi on it inthree days and no more than 1/4 of its surface had fungi after 21 days.Pine treated with (B) had about 1/2 of its surface with fungi in 3 days.Fungi spread on this sample very slowly and it had portions still freeof fungi after 21 days.

Fungi fluorished in the agar microscopically close to the treated wood.These samples showed no zone of inhibition for growth of fungi near thewood.

EXAMPLE 13

An experiment was carried out to determine if multiple washings withvarious liquid media would decrease the activity on a treated substrate.

A commercial cellulose sponge was washed exhaustively in tap water andboiled for 45 minutes in distilled water and dried to constant weight at80° C. Cylinders were cut from the dry sponge with a cork borer. Eachcylinder weighed about 1.05 g. and was about 3/4" in diameter and 11/8"long.

Cylinders were wet with aqueous solutions, about 35 ml. that contained10 mg. of O₃ /₂ Si(CH₂)₃ N(CH₂)₃ C₁₈ H₃₇ ⁺ Cl⁻ salt. The solution wassqueezed and worked into a cylinder which swelled, softened and absorbedmost of the liquid and was then labeled cylinders A. All cylinders weresterilized prior to washing in order to overcome any affect frombacteria in the sponge.

A cylinders were heated to 80° for 0.5 hr. and were labeled cylindersA'.

A and A' cylinders were forced into a glass tube and about 345 liters oftap water/g. of dry sponge was forced thru them during 4 days.

These cylinders were labeled respectively B and B'.

An A cylinder was forced into a glass tube and one liter of homogenizedmilk was forced through it slowly. This was labeled Cylinder C;

An A cylinder was forced into a glass tube and one liter of Gallo brandHardy Burgundy was forced through it slowly. This cylinder was labeledCylinder D;

An A cylinder was forced into a glass tube and one liter of Budweiser®Beer was forced through it slowly and this cylinder was labeled CylinderE;

An A cylinder was forced into a glass tube and one liter of sea waterwas forced through it slowly and this cylinder was labeled Cylinder F.

An untreated cylinder and cylinders A, A', B and B' were inoculated withP. aeruginosa ATCC 870, eluted with four 10 ml. portions of sterilewater, pour plated in letheen growth agar, incubated at 37° and theviable cells in the agar were counted and shown in Table XIII as numberper ml.

Sponge cylinders A, B, C, D, and E were sterilized at 15 psig, 120° for15 minutes in steam. An untreated sponge cylinder and A, B, C, D, E andF were inoculated heavily and tested as above.

                  TABLE XIII                                                      ______________________________________                                        P. aeruginosa Recovered from 1 g. of Sponge                                            No. of viable cells/ml. of culture                                            Sponge        Sponge                                                          Not Sterilized                                                                              Sterilized                                             ______________________________________                                        Control    232,700         2,075,000                                          A             380          186,750                                             A'           200                                                             B          122,000         954,000                                             B'        184,000                                                            C                          934,000                                            D                          352,000                                            E                          581,000                                            F                           62,250                                            ______________________________________                                    

These data indicate that the cellulose sponge became remarkablyantimicrobial treated at a very low level. Heating the sponge had verylittle effect upon its activity. The activity was reduced but remainedhigh after washing exhaustively with diverse liquids.

EXAMPLE 14

Pieces of cloth were treated by the Paint-on technique with (CH₃ O)₃Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ in water to treat the cloth to a level of1% by weight. Some pieces were sterilized after treatment in anautoclave with steam for fifteen minutes.

A piece of Nylon cloth was treated with (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₃ ⁺ Cl⁻to give a level of 0.1% by weight.

The pieces of cloth were tested by AATCC-Test Method 100-1970, forevaluation of Antimicrobial Finishes on Fabrics with a contact time of30 minutes.

                  TABLE XIV                                                       ______________________________________                                        Type of    Type of       % Reduction                                          Fabric     Organism      Initial  Sterilized                                  ______________________________________                                        Cotton     S. aureus     93       92                                                     K. pneumoniae 81.5                                                 Polyester  S. aureus              99.1                                                   K. pneumoniae          68.6                                        Nylon      S. aureus     100                                                  ______________________________________                                    

The MIC of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₃ ⁺ Cl is very high. The compoundshowed no biocidal activity in solution at concentrations as high as 10%by weight against the organisms in Table XIV.

EXAMPLE 15

A colloidal dispersion of solid methylsilsesquioxane (CH₃ SiO₃ /₂)_(n)particles, 10% by weight, was prepared by hydrolyzing an emulsion ofmethyltrimethoxysilane (10 g.) in water (37.1 g.) with 0.75 g. of (CH₃O)₃ Si(CH₂)₃ N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ as an emulsifier and 0.25 ml. of 1 NNH₄ OH as a catalyst for hydrolysis.

A second dispersion was made with (0.75 g.) C₁₂ H₂₅ N(CH₃)₃ ⁺ Cl⁻ as theemulsifier.

Methanol liberated by hydrolysis of the methoxysilanes was removed froma portion of each dispersion by distillation at 90°-95° withoutdisturbing the stability of the colloidal dispersions.

During these preparations the pH of the aqueous phase decreased slowlyfrom pH 11 due to the NH₄ OH, to a pH of 6.5 for the first example andpH 8 in the second. This change in pH indicated the ion-exchangedescribed in Example 3 had occurred between the cationic emulsifyingagents and the surfaces of the colloidal particles.

These were exceedingly small particle size dispersions with theappearance of a slightly hazy, slightly blue liquid.

The standard serial tube dilution test was used with these colloidaldispersions. No agitation was required to keep these surfaces dispersedand Brownian motion was sufficient to cause contact with organisms.

Sample 2 was active at 100 μg/ml. 100 μg. of dispersion could contain nomore than 2 μg. of C₁₂ H₂₅ N(CH₃)₃ ⁺ Cl⁻, so that the apparent MIC forthis substance was decreased 100 to 1000 fold by adsorption on colloidalparticles.

EXAMPLE 16

A colloidal dispersion of methylsilsesquioxane (CH₃ SiO₃ /₂)_(n)particles 5% by weight was prepared in water with 0.7%dodecylbenzyldimethylammonium chloride as the dispersing agent. Thecolloidal particles were about 125 A in diameter.

The serial tube dilution test indicated that the M.I.C. of thiscolloidal dispersion was 100 ppm for E. coli and 1000 ppm for S. aureus.This would correspond to concentrations of the cationic dispersing agentof 0.7 ppm for E. coli and 7 ppm for S. aureus.

The MIC of the dispersing agent in solution is 750 ppm for both E. coliand S. aureus. This experiment indicated that particles so small thatthey move by Brownian motion as colloidal particles in water contact andkill microorganisms. The activity of the cation in this example wasincreased by absorption on the particle by 1000 fold for E. coli and 100fold for S. aureus.

Compare, for example, page 54, Table 22 of Dekker, supra page 3.

EXAMPLE 17

Cotton thread was treated with a 0.5% solution of (CH₃ O)₃ Si(CH₂)₃N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ in water and air dried. The treated thread anduntreated thread were buried in soil inoculated with A. niger ATCC-9642;A. flavus, ATCC 9643; A. versicolor ATCC 11730; P. funiculosum, ATCC9644; C. globosum ATCC 6205 as in Mil. Std. 810B. The tensile strengthof the thread was measured as in ASTM D 1682.

    ______________________________________                                                    Pounds/sq.in.                                                                            Tensile Strength                                                   Initial    After 7 days                                           ______________________________________                                        no treatment  5.1          0.7                                                treated cotton                                                                              5.8          5.6                                                ______________________________________                                    

The treatment very effectively reduced or prevented fungal attack uponcotton.

EXAMPLE 18

Nylon cloth was treated with a 0.1% solution of (CH₃ O)₃ Si(CH₂)₃N(CH₃)₂ C₁₈ H₃₇ ⁺ Cl⁻ in water and air dried. The Nylon was then sprayedwith a culture of Candida albicans ATCC 10231, and the number of viableorganisms recoverable from the cloth was determined.

    ______________________________________                                                Organisms recovered/ml                                                        three runs                                                                              Average    % Reduction                                      ______________________________________                                        no treatment                                                                            200/210/205 205         0                                           treated Nylon                                                                           22/24/18     21        90                                           ______________________________________                                    

Cotton cloth was treated in the same way and tested with Trichophytoninterdigitale ATCC 9533.

    ______________________________________                                        % surface growth in 28 days                                                                         Average %                                               ______________________________________                                        no treatment   100/100/100  100                                               treated cotton 20/20/10      17                                               ______________________________________                                    

A polyester-cotton cloth was treated with a 0.33% solution in the sameway and sprayed with cultures of the following organisms and the numbersrecoverable were measured.

    ______________________________________                                                  No. of Organisms/ml after 6 hrs.                                    Organism   Untreated  Treated   % Reduction                                   ______________________________________                                        Micrococcus sp.                                                                          215,500    2,700     99                                            Staph. epidermidis                                                                       58,000     3,000     95                                            Enterobacter-                                                                 aglomerans 1,355,000  16,500    90                                            Acinetobacter-                                                                calcoaceticus                                                                            3,500      1,000     72                                            Micrococcus sp.                                                                          305,000    0         100                                                      395,000    200       99                                            Staph. aureus                                                                            200,000    200       99                                            ______________________________________                                    

EXAMPLE 19

Aerosil-200 silica manufactured by the Degussa Co., West Germany wastreated in aqueous solution by mixing (CH₃ O)₃ Si(CH₂)₃ SC(NH₂)⁺ ₂ Cl⁻into the water and adding the Aerosil-200 to the mixture. It washomogenized for 5 minutes using a blender. The material was then driedat 90° C. in an oven for 10-15 minutes. The silica was then formulatedinto a cold cream in the following manner. Two parts were prepared.

    ______________________________________                                        Part A-   2.5 gms Stearyl Alcohol                                                       4.0 gms White petrolatum                                                      0.5 gms Sorbitan Monooleate                                                   2.5 gms Isopropyl Myristate                                         Part B-   Propylene glycol                                                                              3.5 gms                                                       *Polyoxyl-40 Stearate                                                                         1.25 gms                                                      Water          35.8 gms                                                       Preservative Powder                                                                          5% powder based on                                             Prepared Above total weight of B                                                             with 1% active                                                                perservative thereon                                 ______________________________________                                         *Polyoxyl-40 Stearate is a polyethylene oxide stearate ester having 40        CH.sub.2 CH.sub.2 O units per stearic acid residue.                      

The parts A and B were heated separately to 75° C. and then B was addedto A with agitation until the material became creamy and smooth. Thematerial was slowly cooled with stirring until it began to set up andthen it was allowed to cool to room temperature without stirring.

The cold cream was evaluated by a method set forth in CTFA CosmeticJournal, Vol. 5, No. 1, "Evaluation of Methods for DeterminingPreservative Efficacy". Briefly, aliquats of the cream were challengedwith the organisms and any growth was observed at day intervals duringwhich time the challenged plates were incubated at 37° C. The results ofthis test can be found on Table XV.

                                      TABLE XV                                    __________________________________________________________________________    "Evaluation of Treated Silica in Cold Cream Formulation"                      CFTA Challenge Test - Challenge Organism-                                     Pseudomonas Aeroginosa ATCC 15442                                             Bacterial Count                                                               Sample Day 0 Day 1 Day 7                                                                              Day 14                                                                             Day 21                                                                             Day 28                                      __________________________________________________________________________    control                                                                       (no treated                                                                   powder)                                                                              7,250,000                                                                           10,200,000                                                                          615,000                                                                            450,000                                                                            500,000                                                                            1,825,000                                   5.0% powder                                                                   with 1.0%                                                                     active                                                                        compound                                                                               91,500                                                                            <10   <10  <10  <10  <10                                         __________________________________________________________________________

That which is claimed is:
 1. A method of reducing the number of viablebacteria, fungi, algae and yeast in media by physically contacting thebacteria, fungi, algae and yeast with a surface which has been alteredin a manner which comprises contacting a substrate, which develops anegative charge in water, with an amount effective to inhibit the growthof said microorganisms of a substance that ionizes in water to formcations and anions which substance is selected from the group consistingof sulfonium salts of the formula R⁸ R⁹ R¹⁰ S⁺ X⁻, sulfonium salts ofthe formula ##STR6## isothiuronium salts of the formula RSC(NH₂)₂ ⁺ X⁻,isothiuronium salts of the formula ##STR7## phosphonium salts of theformula R⁴ R⁵ R⁶ R⁷ P⁺ X⁻, and phosphonium salts of the formula ##STR8##wherein R⁸, R⁹ and R¹⁰ are independently alkyl groups or aralkyl groupswherein there is a total of less than 30 carbon atoms in the molecule ora silylsubstituted alkyl radical of the formula ##STR9## where Y is ahydrolyzable group, Q is an alkyl radical of 1 or 2 carbon atoms or(CH₃)₃ SiO, a has an average value of 0-3, b has an average value of0-3, n is an integer of 1 or greater and X⁻ is a water solublemonovalent anion, R is independently alkyl or aralkyl groups whereinthere is a total of less than 20 carbon atoms in the molecule or asilylsubstituted alkyl radical of the formula ##STR10## as definedabove, R⁴, R⁵, R⁶ and R⁷ are as defined above for R⁸, R⁹ and R¹⁰ ; R¹¹and R¹² are independently alkyl groups or aralkyl groups wherein thereis a total of less than 60 carbon atoms in the molecule and d is aninteger of 1 or greater.
 2. The method of claim 1 in which themicroorganisms are gram-positive or gram-negative bacteria.
 3. Themethod of claim 1 in which the microorganisms are fungi.
 4. The methodof claim 1 in which the microorganisms are yeasts.
 5. A method asclaimed in claim 1 wherein the sulfonium salt is a sulfonium halide. 6.A method as claimed in claim 5 wherein the halide is chloride.
 7. Amethod as claimed in claim 5 wherein the halide is iodide.
 8. A methodas claimed in claim 5 wherein the halide is bromide.
 9. A method asclaimed in claim 1 wherein in the groups R⁸, R⁹ and R¹⁰, at least onesuch group has at least 10 carbon atoms.
 10. A method as claimed inclaim 5 wherein the sulfonium salt is (CH₃ O)₃ Si(CH₂)₃ S⁺ (CH₃)C₁₈ H₃₇I⁻.
 11. A method as claimed in claim 5 wherein the sulfonium salt is(CH₃ O)₃ Si(CH₂)₃ S⁺ (CH₃)C₂ H₅ I⁻.
 12. A method as claimed in claim 1wherein the isothiuronium salt is an isothiuronium halide.
 13. A methodas claimed in claim 12 wherein the isothiuronium salt is anisothiuronium chloride.
 14. A method as claimed in claim 12 wherein theisothiuronium salt is an isothiuronium iodide.
 15. A method as claimedin claim 12 wherein the isothiuronium salt is an isothiuronium bromide.16. A method as claimed in claim 12 wherein the isothiuronium salt is(CH₃ O)₃ Si(CH₂)₃ S⁺ C(NH₂)₂ Cl⁻.
 17. A method as claimed in claim 1wherein the phosphonium salt is a phosphonium halide.
 18. A method asclaimed in claim 17 wherein the phosphonium salt is a phosphoniumchloride.
 19. A method as claimed in claim 17 wherein the phosphoniumsalt is phosphonium iodide.
 20. A method as claimed in claim 17 whereinthe phosphonium salt is a phosphonium bromide.
 21. A method as claimedin claim 17 wherein the phosphonium salt is (CH₃ O)₃ Si(CH₂)₃ P⁺ (n-C₄H₉)₃ I⁻.
 22. A method as claimed in claim 21 wherein the phosphoniumsalt is (CH₃ O)₃ Si(CH₂)₃ P⁺ (C₆ H₅)₃ I⁻.
 23. A method as claimed inclaim 1 wherein the sulfonium salt is a sulfonium carboxylate.
 24. Amethod as claimed in claim 1 wherein the isothiuronium salt is anisothiuronium carboxylate.
 25. A method as claimed in claim 1 whereinthe phosphonium salt is a phosphonium carboxylate.
 26. A method ofreducing the number of viable bacterial, fungi, algae and yeast in mediaby physically contacting the bacteria, fungi, algae and yeast with asurface which has been altered in a manner which comprises contacting asubstrate, which develops a negative charge in water, with an amounteffective to inhibit the growth of said microorganism of a substancethat ionizes in water to form cations and anions which substanceconsists of an organic amine of the formula ##STR11## in which d in eachcase is an integer of 1 or greater.